Gla is an unusual amino acid formed by γ-carboxylation of a limited number of glutamic acid (Glu) residues in certain proteins. Carboxylation, which is an early posttranslational event, requires vitamin K and the presence of Gla in proteins/peptides has so far always been associated with the action of vitamin K (Furie et al., 1999; Suttie, 1985).
Carboxylation is catalysed by a vitamin K-dependent carboxylase, an enzyme that has been purified and cloned. The complementary DNA has been expressed and the recombinant protein found to be fully active. An unstable metabolite of vitamin K, derived from the hydroquinone form of the vitamin, is involved in the abstraction of a proton from the γ-carbon atom of certain peptide-bound Glu residues with subsequent incorporation of CO2 and formation of Gla, a malonic acid derivative. Typically, Gla-containing proteins have an N-terminal portion termed the propeptide that is recognised by the carboxylase. Following carboxylation and release of the modified protein from the enzyme the propeptide is removed by limited proteolysis.
Proteins that contain Gla are often referred to collectively as the ‘vitamin K-dependent proteins’ (Stenflo and Dahlbäck, 1994). Among these proteins the hemostatic factors that require vitamin K for normal synthesis have been studied in most detail. They comprise prothrombin (factor II), factor VII, factor IX, factor X and the two regulatory proteins, protein C and its cofactor protein S.
Structurally related to factors VII, IX, X and protein C is protein Z that, according to recent investigations, may be involved in the regulation of blood coagulation.
Homologues to the proteins of this group have been found in numerous vertebrates including snakes, e.g. the venom of the toxic snake Tropidechis carinatus contains a protein that resembles factor X both in terms of structure and biological activity (Joseph et al., 1999). Growth arrest-specific protein 6 (Gas6) is a homologue of protein S that seems to be involved in apoptosis.
The Gla-containing hemostatic factors and their homologues all have an amino-terminal (N-terminal) Gla domain of approximately 45 amino acid residues in which all 9-12 Glu residues contained therein are normally carboxylated to Gla. The propeptide, which is encoded on the same exon as the N-terminal part of the Gla domain, is recognised by the vitamin K-dependent carboxylase and activates the enzyme. After binding its substrate, the carboxylase seems to carboxylate all Glu residues in the vicinity of the propeptide (i.e. up to about residue +45 in the vitamin K-dependent hemostatic factors) irrespective of the surrounding amino acid sequence.
The vitamin K-dependent coagulation factors are primarily synthesised in the liver and can be divided into three groups depending on their domain structure. Factors VII, IX and X and protein C have an N-terminal Gla domain that is followed by two domains that are homologous to the epidermal growth factor (EGF) whereas the C-terminal part of the molecule is occupied by a serine proteinase domain. Another homologue, protein Z, also has this domain structure. However, natural mutations in the protein Z gene have altered the active site residues of the protein and it has no proteinase activity. Prothrombin also has an N-terminal Gla domain that is followed by a characteristic loop, two so called kringle domains, and a serine proteinase domain. Protein S and Gas6 are homologues. In protein S, the region that follows the Gla domain contains one or two peptide bonds that are particularly sensitive to thrombin. This region is followed by four EGF-like domains, whereas the C-terminal half of the molecule is homologous to a sex steroid-binding protein in human plasma.
The vitamin K-dependent proteins that are involved in blood coagulation and its regulation are, with the exception of protein S and protein Z, proenzymes of serine proteinases. Although the enzymes have full activity against substrates with small molecular masses (e.g. short peptides), their activity is minimal against their physiological substrate(s) (for factor VIIa these are factors IX and X; for factor IXa, factor X; for factor Xa, prothrombin; the ‘a’ in VIIa and so forth denotes the enzymatically active form of the protein) unless they are in complex with their appropriate membrane-bound cofactor, thus forming enzymatically active membrane-bound macromolecular complexes. The enzyme in the complex, for example factor Xa in complex with its cofactor, factor Va (here the ‘a’ denotes the active form of the cofactor to distinguish it from the inactive pro-cofactor) activates its substrate, prothrombin, by limited proteolysis.
The nine to twelve Gla residues in the Gla domain mediate binding of 7-10 calcium ions which is crucial to keep the Gla domain in the conformation that is required for biological activity and hence for its interaction with biological membranes.
Clinically used anticoagulant drugs such as Warfarin® function by inhibiting Gla formation in the vitamin K-dependent proteins, thus reducing the calcium affinity of the protein and its affinity for biological membranes. Such drugs are used, for instance, in the treatment of venous thrombosis and pulmonary embolism.
Gla has also been found in two proteins in mineralised tissues; bone Gla protein (or osteocalcin) and matrix Gla protein. These proteins are smaller than the coagulation factors and contain fewer Gla residues. However, they have structures resembling the propeptide that are crucial for substrate recognition by the vitamin K-dependent carboxylase. These carboxylations have also been shown to have an obligatory requirement for vitamin K. Bone Gla protein is involved in the regulation of calcification of mineralised extracellular matrices and matrix Gla protein seems to have a broad role in calcium homeostasis.
In addition to coagulation factors and the two proteins found in mineralised tissues, the presence of Gla has been reported in several proteins in urine, kidney, placenta, the chorioallantoic membrane of chicken eggs and so forth but in most cases these proteins have not been characterised in molecular detail (Vermeer and De Boer-Van den Berg, 1985).
More recently, the cDNA of two putative membrane proteins with typical Gla domains (hence resembling the coagulation factors) were cloned (Kulman et al., 1997). The function of these proteins is not yet known nor have they been characterised at the protein level.
The discovery of Gla in the venom of predatory snails of the genus Conus and the finding that vitamin K is involved in its biosynthesis in these molluscs indicates that vitamin K presumably has important functions in many more biological systems than hitherto assumed (Olivera et al., 1990). Moreover, the biological activity of a Conus-derived toxin was shown to depend on the presence of Gla, i.e. a synthetic toxin with the same structure except that Gla had been substituted with Glu was biologically inactive.
From the above it should be obvious that the identification of Gla in proteins is of considerable interest, and that this interest is rapidly increasing as a result of recent discoveries implicating Gla in the function of novel proteins with important biological functions. This field of research would be much simplified were there rapid, simple, dependable and inexpensive methods to identify Gla in proteins.
The standard method for the identification and quantification of amino acids has been amino acid analysis after acid hydrolysis. However, this method is unsuitable for the identification of Gla as it is a malonic acid derivative and hence is decarboxylated to Glu by acid hydrolysis. Therefore, alkaline hydrolysis has been used to quantify Gla in proteins. Although suitable for quantification of Gla in purified proteins, this method is of limited use for the identification of Gla-containing proteins in biological fluids or tissue extracts.
As the Gla-containing proteins that have been studied in detail have been found to bind calcium, ‘calcium blotting’ has sometimes been used to identify Gla-containing proteins. The method entails SDS-polyacrylamide gel electrophoresis followed by transfer to a suitable membrane (e.g. nitrocellulose membrane) that is then incubated in a buffer containing a radioactive calcium isotope (45Ca). After drying and exposure of the membrane to a suitable film or detector, the calcium-binding protein(s) can often be identified. The weaknesses of the method are that it does not discriminate between Gla-containing and other calcium-binding proteins and it is not quantitative. Moreover, a high calcium affinity is required for the protein to be detected. Finally, the method requires the protein to retain a native, calcium-binding conformation despite having been exposed to SDS.
Colourimetric methods for the detection of Gla-containing proteins have been devised but they have not been widely used (Jie et al., 1995; Nishimoto, 1990).
During protein/peptide sequencing (using Edman chemistry) Gla is easily identified after chemical modification (Cairns et al., 1991). However, this requires homogeneous proteins and should be preceded by a quantitative Gla measurement of an alkaline hydrolysate of the protein.
Mass spectrometry is an indispensable method for the characterisation of homogeneous Gla-containing proteins/peptides (e.g. Rigby et al., 1999). However, despite its merits, mass spectrometry is not suitable for the detection of Gla-containing proteins in biological fluids or tissue extracts.
A simple immunochemical method for the detection of Gla in biological fluids and tissue specimens is needed, as well as a simple method for the affinity purification of Gla-containing proteins. Such a method would lead to new advances in research relating to vitamin K-dependent proteins and would also be of considerable use in the purification of therapeutically useful recombinant vitamin K-dependent proteins such as coagulation proteins. Such methods have been devised for the simple identification and purification of proteins containing phosphorylated tyrosine, serine and threonine residues and have turned out to be very useful (e.g. Frackelton et al., 1983).